Multi-layered microfibers and use thereof

ABSTRACT

Multi-layered electrospun microfibers and/or microtubes, comprising cells and/or molecules of interest. The invention is further directed to compositions comprising said microfibers and/or microtubes and methods of use thereof in various applications.

FIELD OF INVENTION

The present invention is directed to, inter alia, multi-layered electrospun microfibers and/or microtubes, comprising cells and/or molecules of interest. The invention is further directed to 5 compositions comprising said microfibers and/or microtubes and methods of use thereof in various applications.

BACKGROUND OF THE INVENTION

International application publication No. WO 2008/041183 provides electrospun 10 microtubes and a method of producing a microtube, comprising co-electrospinning two polymeric solutions through co-axial capillaries to thereby produce the microtube, wherein a first polymeric solution of the two polymeric solutions is for forming a shell of the microtube and a second polymeric solution of the two polymeric solutions is for forming a coat over an internal surface of the shell, the first polymeric solution is selected solidifying faster than the second polymeric 15 solution and a solvent of the second polymeric solution is selected incapable of dissolving the first polymeric solution.

International application publication No. WO 2009/104174 provides a method of attaching a cell or a membrane-coated particle-of-interest to a microtube. International application publication No. WO 2009/104176 provides a method of attaching a cell or a molecule-of-interest 20 to a microtube.

There is a need for improved electrospun cell-bearing microtubes and/or microfibers with increased cell viability and density.

SUMMARY OF THE INVENTION

According to one aspect, the present invention provides an electrospun concentric multi-layered microfiber comprising an exterior layer, an interior layer, and an intermediate layer residing between the exterior layer and the interior layer, said intermediate layer comprises one or more water soluble polymers, and said interior layer comprises one or more types of living cells and/or molecules of interest, and optionally one or more water soluble polymers and/or oligomers. 30

According to another embodiment, there is provided a concentric multi-layered electrospun microfiber comprising an exterior layer, an interior layer, and an intermediate layer residing between the exterior layer and the interior layer, said intermediate layer comprises one or more water soluble polymers, and said interior layer comprises cells and/or molecules of interest.

According to another embodiment, there is provided a concentric multi-layered electrospun 5 microfiber of the present invention, wherein interior layer further comprises water soluble polymers or oligomers.

According to another embodiment, there is provided a triple-layer microfiber.

According to another embodiment, said microfiber is hollow.

According to another embodiment, said interior layer, intermediate layer and/or interior 10 layer are substantially porous layers.

According to another embodiment, said water soluble polymers of said intermediate layer are selected from natural polymers, synthetic polymers or combinations thereof.

According to another embodiment, said exterior layer comprises one or more organic soluble polymers. According to another embodiment, said organic soluble polymer of said exterior 15 layer is selected from the group consisting of: poly(vinylidenefluoride), poly (e-caprolactone) (PCL), polyamide, poly(siloxane), poly(silicone), poly(ethylene), poly(2-hydroxy ethylmethacrylate), poly(methyl methacrylate), poly(vinyl acetate), polyacrylamide, poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polylactide, polyglycolide, poly(lactide-co-glycolide), polyanhydride, polyorthoester, poly(carbonate), poly(acrylo nitrile), polyaniline, polyvinyl carbazole, polystyrene, poly(vinyl phenol), polyhydroxyacid, poly(caprolactone), polyanhydride, polyhydroxyalkanoate, polyurethane, collagen, cellulose, and combination/copolymers thereof.

According to another embodiment, said organic-soluble polymer is poly(vinylidenefluoride-co-hexafluor-opropylene) (PVDF-HFP).

According to another embodiment, said exterior layer comprises water-soluble polymers. 25 According to another embodiment, said exterior layer comprises water soluble polymers selected from the group consisting of: poly(vinyl pyrrolidone), poly(N-vinyl pyrrolidone), poly(vinyl alcohol), poly(acrylic acid), poly(ethylene glycol), poly(ethylene oxide), albumin, alginate, chitosan, starch, hyaluronic acid, albumin, alginate, chitosan, starch, polypetides and combination/copolymers thereof.

According to another embodiment, said exterior layer further comprises antifouling agents.

According to another embodiment, said water soluble polymers of said intermediate layer are selected from the group consisting of poly(acrylic acid), poly(vinyl pyrrolidone), poly(N-vinyl pyrrolidone), poly(vinyl alcohol), poly(acrylic acid) poly(ethylene glycol), poly(methacrylic acid), poly(ethylene oxide), polyhydroxyacid, alginate, starch, chitosan, dextran, albumin, chitosan, hyaluronic acid, polyppertides and combination/copolymers thereof.

According to another embodiment, said water soluble polymers and/or oligomers of said interior layer are selected from natural and synthetic polymers and/or oligomers, or combinations thereof.

According to another embodiment, said water soluble synthetic polymer is selected from the group consisting of PVA, PVP, PEO, PAA, polystyrene sulfonate and combinations or copolymers thereof.

According to another embodiment, said water soluble natural polymer is selected from the group consisting of polysaccharides (e.g. alginate), starch, hyaluronic acid, alginate, dextran, chitosan and combinations or copolymers thereof.

According to another embodiment, said oligomer is selected from the group consisting of chitosan oligomeric derivatives, and saccharides oligomers.

According to another embodiment, the cell concentration within said internal layer or core is at least 10 OD.

According to another embodiment, said interior layer further comprises media for maintaining and/or supporting said cell viability and/or activity.

According to another embodiment, said molecules of interest are selected from hormones, growth factors, enzymes, nutrients and shock resistance molecules.

According to another embodiment, said exterior layer has a thickness of about 5 nm to 10 micrometer.

According to another embodiment, said intermediate layer has a thickness of about 5 nm to 10 micrometer.

According to another embodiment, said internal layer has a thickness of about 5 nm to 10 micrometer.

According to another embodiment, said interior layer has a diameter of at most 50 micrometer.

According to another aspect, there is provided a method for producing a concentric triple-layered microfiber, the method comprising providing a first polymeric solution for forming an exterior layer of the microfiber, a second polymeric solution for forming an intermediate layer of the microfiber, and a third solution for forming an interior layer or a core of the microfiber, said third solution comprises cells; and co-electrospinning the first polymeric solution, second polymeric solution and the third solution through a triple-axial capillaries, thereby producing the concentric triple-layered microfiber.

According to another embodiment, the first polymeric solution solidifies faster than the second polymeric solution.

According to another embodiment, the second polymeric solution solidifies faster than the third solution.

According to another embodiment, the second polymeric solution and said third solution comprise the same solvent.

According to another embodiment, the second polymeric solution comprises a solvent which is incapable of dissolving the first polymeric solution.

Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a spinneret with three co-axial capillaries.

FIGS. 2A-C illustrate triple-layered microfibers and/or microtubes according to some embodiments of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides, in one embodiment, concentric multi-layered electrospun microfibers or microtubes comprising cells and/or any biological elements. The invention further provides, in another embodiment, compositions, kits and systems comprising said microfibers and methods of use thereof In another embodiment, there is provided a method of fabricating said microfibers.

The present invention is based, in part, on the surprising finding that a concentric multi-layered electrospun microfiber enables the incorporation of extremely high concentration of viable cells in the microfiber's internal layer or core. In an exemplary embodiment, the microfibers disclosed herein were shown to incorporate viable cells at an extremely high concentration of about 50 OD (equivalent to about 10¹¹ cells/ml).

The electrospun microfibers of the invention comprise an intermediate layer which acts as a barrier between the exterior layer (which comprises, in some embodiment, solvent harmful to cell or other biologics) and the interior layer comprising said cells. The incorporation of an intermediate layer between the exterior layer and the interior layer advantageously allows incorporation of high concentration of viable cells and/or biologics. In some embodiments, the solvent selected for forming the exterior layer of the microfiber is an organic-based solvent and the solvent selected for forming the intermediate and/or internal layer of the microfiber is a solvent compatible for cell growth and viability.

According to another embodiment, the present invention provides a multi-layered concentric electrospun microfiber or microtube comprising: (i) an exterior layer comprising one or more polymers, (ii) an interior layer or a core, and (iii) an intermediate (i.e., middle) layer situated between the exterior layer and the interior layer, said middle layer comprises one or more water-soluble polymers. In one embodiment, the interior layer comprises a cell suspension, and/or a suspension of molecules of interest. In another embodiment, the interior layer comprises one or more water soluble polymers and/or oligomers and cells and/or molecules of interest.

As used herein, the term “concentric” means that all layers of the multi-layer microfiber have a substantially common center, sharing a co-axial orientation. As such, concentric indicates that intermediate channels surround the core channel and, in turn, the outer channel surrounds the intermediate channels. In an embodiment, all the channels may share the same geometric center or axis.

As used herein, “intermediate” indicates that a layer is disposed between the core region and the outer layer. The outer layer will typically encompass the whole of the core region, at least in a cross section of the body, and the intermediate layer or layers will be disposed between the outer layer and the core region.

Reference is now made to FIGS. 2A, 2B, and 2C, illustrating a simplified, schematic illustration of a cross-section view of a portion of a multi-layer microfiber of the invention. In another embodiment, the microfiber is a non-hollow fiber, comprising (or composed of) an external layer 20 (e.g., comprising organic-soluble polymers), an intermediate layer 20 optionally comprising water-soluble polymers, and an internal layer 40 encapsulating cells and/or molecules of interest 10. Said internal layer 40 optionally comprises water-soluble polymers, as described herein.

Reference is made to FIG. 2A, illustrating a simplified, schematic illustration of a cross-section view of a portion of a triple-layer microfiber 200, constructed and operative in accordance with another non-limiting embodiment of the present invention.

The term “non-hollow”, as used herein, refers to an internal layer substantially filling the inner diameter of the fiber (i.e., the microfiber's core). In another embodiment, said microfiber is at least a triple-layered microfiber. In another embodiment, said microfiber is a triple-layered microfiber.

In some embodiment, said microfibers are substantially hollow, e.g., forming microtubes. In one embodiment, there is provided a multi-layered concentric electrospun microtube. As used herein, the term “microtube” refers to a hollow tube having an inner diameter of e.g., at most 50 μm, which is devoid of an electrospun polymer. In another embodiment, said microtube is at least a triple-layered microtube.

In one embodiment, said multi-layer microtube comprises an external layer (e.g., comprising organic-soluble polymers), an intermediate layer comprising water-soluble polymers, and an internal layer comprising water-soluble polymers and encapsulated cells and/or molecules of interest. Reference is made to FIG. 2B, illustrating a simplified, schematic illustration of a cross-section view of a portion of a multi-layer microfiber 300, constructed and operative in accordance with another non-limiting embodiment of the present invention. It should be understood, that within such multi-layer microtube, cells may be entrapped in the internal layer of the microtube and/or in the internal lumen of the tube.

In one embodiment, said multi-layer microtube comprises an external layer (e.g., comprising organic-soluble polymers), an intermediate layer comprising water-soluble polymers, and an internal layer comprising cells and/or molecules of interest. Reference is made to FIG. 2C, illustrating a simplified, schematic illustration of a cross-section view of a portion of a multi-layer microfiber 400, constructed and operative in accordance with another non-limiting embodiment of the present invention. The multi-layer microfiber 400 a double layered hollow microtube of the invention comprising an external layer 20, and intermediate layer 30 and a third internal solution 40 comprising cells 10 and/or molecules of interest suspended in a liquid 10 (e.g., water, cell media, saline, etc.), filling the lumen of the tube without deposing of a layer.

According to one embodiment, said internal layer comprising cells is formed from an unspinnable solution. As used herein, the term “unspinnable solution” refers to a solution which does not undergo stretching and elongation under an electrospinning process. The spinnability of a solution may be determined by one skilled in the art, such as by the viscoelasticity of the solution (e.g., according to the polymeric concentration and/or molecular weight within the solution).

According to some embodiments, said intermediate layer is formed from an unspinnable solution so as to form a viscous layer (i.e., a layer which did not undergo stretching and elongation under a typical electrospinning process. In another embodiment, the intermediate layer of the microfiber disclosed herein is substantially devoid of cells. An intermediate layer substantially devoid of cells refers to, in some embodiments, a layer being electrospun using a polymeric solution which is devoid of cells.

The multi-layered microfiber and/or microtube of the invention are typically processed under a single-step process, i.e., a single electrospinning step. In another embodiment, the multi-layered microfiber and/or microtube of the invention are processed by co-electrospinning three or more solutions through triple-axial or multi-axial capillaries, respectively. Methods of electrospinning the microfibers of the invention are further detailed herein below.

In one embodiment, said intermediate layer allows diffusion of the cells therethrough. In another embodiment, said intermediate layer substantially prevents diffusion of the cells therethrough.

In another embodiment, said intermediate layer is substantially porous and/or non-continuous. In another embodiment, said porous layer comprises pores suitable for diffusion of nutrients and gasses (e.g., oxygen) to and from the encapsulated cells within the inner layer of said microfiber and/or microtube. In one embodiment, said porous layer comprises pores of about 1 nm or more.

In another embodiment, said polymers of said intermediate layer are selected from natural polymers, synthetic polymers or combinations thereof. In another embodiment, said intermediate layer comprises one or more water-soluble polymers. In another embodiment, said intermediate layer is substantially devoid of cells.

In one embodiment, said polymers of said exterior layer are substantially biodegradable. In another embodiment, said polymers of said exterior layer are substantially not biodegradable.

In another embodiment, said exterior layer comprises one or more organic soluble polymers. None limiting examples of organic-soluble polymers of said exterior layer include poly(vinylidenefluoride (PVDF), poly (e-caprolactone) (PCL), polyamide, poly(siloxane), poly(silicone), poly(ethylene), poly(2-hydroxy ethylmethacrylate), poly(methyl methacrylate), poly(vinyl acetate), polyacrylamide, poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polylactide, polyglycolide, poly(lactide-coglycolide), polyanhydride, polyorthoester, poly(carbonate), poly(acrylo nitrile), polyaniline, polyvinyl carbazole, polystyrene, poly(vinyl phenol), polyhydroxyacid, poly(caprolactone), polyanhydride, polyhydroxyalkanoate, polyurethane, collagen, cellulose, and combination/copolymers thereof. One skilled in the art will appreciate that other organic soluble polymers may be used to form the microtubes and/or microfibers of the invention.

In some embodiments, there is provided polymer selected from the group consisting of a polyvinylidene fluoride, a copolymer, or a terpolymer thereof containing a major portion of vinylidene fluoride. In some embodiments, the polymer comprises polyvinylidene fluoride and at least one copolymerizable monomer selected from the group consisting of hexafluoropropylene, trifluoroethylene, chlorotrifluoroethylene, vinylfluoride and tetrafluoroethylene and any mixture of the homopolymer, copolymer, and terpolymer. In some embodiments, said organic-soluble polymer is poly(vinylidenefluoride-co-hexafluor-opropylene) (PVDF-HFP).

In another embodiment, said exterior layer further comprises antifouling agents.

In another embodiment, said exterior layer comprises one or more water-soluble polymers. In another embodiment, said interior layer comprises one or more water-soluble polymers. In another embodiment, said intermediate layer comprises one or more water-soluble polymers.

None limiting examples of said water soluble polymers include poly(acrylic acid), poly(vinyl pyrrolidone), poly(N-vinyl pyrrolidone), poly(vinyl alcohol), poly(acrylic acid) poly(ethylene glycol), poly(methacrylic acid), poly(ethylene oxide), polyhydroxyacid, alginate, starch, chitosan, dextran, albumin, hyaluronic acid, polypeptides and combination and/or copolymers thereof. One skilled in the art will appreciate that other water soluble polymers may be used to form the microtubes and/or microfibers of the invention.

In another embodiment, said water soluble polymers or oligomers of said interior layer are selected from natural polymers, synthetic polymers or combinations thereof.

In another embodiment, said water soluble synthetic polymer is selected from the group consisting of poly(vinyl alcohol) (PVA), Polyvinylpyrrolidone (PVP), poly(ethylene oxide) (PEO), and Poly(acrylic acid) (PAA).

In another embodiment, said water soluble natural polymer is selected from the group consisting of polysaccharides (e.g. alginate, dextran, starch, hyaluronic acid), proteins (e.g. albumin).

In another embodiment, said oligomers are selected from the group consisting of chitosan oligomeric derivatives.

In another embodiment, said internal layer comprises cell suspension and/or membrane-coated particles (e.g. liposomes) and/or biological elements including, but not limited to, enzymes, hormones, growth factor, salts, nutrients, preserving molecules, media and/or nutrients for maintaining and/or supporting said cell viability.

In another embodiment, said interior layer and/or said intermediate layer further comprises one or more agents selected from nutrients, shock resistance molecules (e.g. glycerol, trehalose, P, N, salts).

In another embodiment, said exterior layer has a thickness of about 5 nm to one or more (e.g., 10) micrometers. In another embodiment, said intermediate layer has a thickness of about 5 nm to 10 micron. In another embodiment, said interior layer has a thickness of about 5 nm to 50 micrometer. In another embodiment, said interior layer has a diameter of at most 50 micrometer.

Cells

As used herein the term “cell” refers to a eukaryotic or prokaryotic cell. According to some embodiments of the invention, the cell comprises a cell wall. Non-limiting examples of cells which comprise a cell wall and which can be attached to the microfiber of the invention include plant cells, bacteria (e.g., Gram positive and Gram negative bacteria), archaea, protozoa, fungi (yeast), and algae. In another embodiment, said cell is an animal cell such as human cells.

According some embodiments, said second solution comprises cells at a concentration of at least 15 Optical Density, at least 20 Optical Density, at least 25 Optical Density, at least 30 Optical Density, at least 35 Optical Density, at least 40 Optical Density, at least 45 Optical Density or at least 50 Optical Density. According some embodiments, said third solution comprises cells at a concentration of at least of at least 3*10̂10 cells/ml.

According to some embodiments of the invention, the cell and/or the molecule of interest is a membrane-coated particle-of-interest such as a liposome or micelles.

According to some embodiments of the invention, the cell comprises a dermis (e.g., insect cells).

According to some embodiments of the invention, the cell has a diameter from about 500 nanometers to about 50 microns, e.g., from about 1-10 microns.

None limiting examples of cells include Chromobacterium violaceum, Pseudomonas maltophila, Pseudomonas aeruginosa, Spirulina planensis, Staphylococcus aureus, Bacillus cereus, Bacillus subtilis and Escherichia coli.

Typically, the cells may be attached to the polymer(s) comprised in the microfiber via covalent or non-covalent binding (e.g., via an electrostatic bond, a hydrogen bond, a van-Der Waals interaction) so as to obtain an absorbed, embedded or immobilized cell to the inner layer of the microfiber of the invention.

According to some embodiments of the invention, covalent attachment of the cell can be via functional groups such as SH groups, amino groups, carboxyl groups which are added to the polymer(s) forming the microfiber.

According to some embodiments of the invention, the cells adhere to the inner layer of the microfiber by use of cell adhesion promoters such as fibronectin.

Use of the Microfibers

In one embodiment, the microfibers and microtubes or compositions comprising same are used in various applications including but not limited to water purification, detoxification, bio-chemicals fermentations, mineral enrichment, tissue grafts and cell-based therapy.

According to an aspect of some embodiments of the present invention there is provided a method of bioremediation, the method comprising contacting a solution containing a contaminant with the microfiber or microtube of some embodiments of the invention, wherein the cell, a portion of the cell or is capable of degrading or assimilating the contaminant.

According to an aspect of some embodiments of the present invention there is provided a method of depleting a molecule from a solution, comprising contacting the solution with the microfiber or microtube of some embodiments of the invention, wherein the molecule is capable of binding to or being processed by the cell, thereby depleting the molecule from the solution.

According to an aspect of some embodiments of the present invention there is provided a method of isolating a molecule from a solution, comprising: (a) contacting the solution with the microfiber or microtube of some embodiments of the invention under conditions which allow binding of the molecule to the cell, and; (b) eluting the molecule from the microfiber or the microtube; thereby isolating the molecule from the solution.

According to an aspect of some embodiments of the present invention there is provided a method of detecting a presence of a molecule in a sample, comprising: (a) contacting the sample with the microfiber or microtube of some embodiments of the invention, wherein the cell is capable of binding to or processing the molecule, and; (b) detecting the binding or the processing; thereby detecting the presence of the molecule in the sample.

Water purification usually entails the removal of toxic chemicals such as mercury, mercurial compounds, and cadmium or elements such as toluene, chloroform, benzene, pesticides and herbicides. In one embodiment, said cell is Pseudomonads. In another embodiment, said Pseudomonads is used to degrade toluene, benzene, phenol, naphthalene, atrazine, and/or certain hydrocarbons from oil.

In another embodiment, the microfibers or compositions comprising same are used in water systems for removal of toxic heavy metals such as cadmium and mercury. None limiting examples of bacterial strains useful for such application include Chromobacterium violaceum, Pseudomonas maltophila, Pseudomonas aeruginosa, Spirulina planensis, Staphylococcus aureus, Bacillus cereus, Bacillus subtilis and Escherichia coli.

According to an aspect of some embodiments of the present invention there is provided a controlled release mechanism, such as that each layer of said microfiber or microtube provides a separate phase of sustained release.

In another embodiment, the microfibers or compositions comprising same are used in fermentation methods.

Production Methods

According to another aspect, there is provided a method for producing the microfibers or microtubes of the invention, the method comprising co-electrospinning three solutions through triple-axial capillaries.

Reference is made to FIG. 1, illustrating a triple-axial capillary (e.g., a spinneret with three co-axial capillaries) used for the triple axial co-electrospining procedure for forming a concentric triple-layer electrospun microfiber or microtube of the invention.

According to some embodiments, there is provided a method for producing a concentric triple-layered microfiber, the method comprising providing a first polymeric solution for forming an exterior layer of the microfiber, a second polymeric solution for forming an intermediate layer of the microfiber, and a third solution for forming an interior layer or a core of the microfiber, said third solution comprises cells; and co-electrospinning the first polymeric solution, second polymeric solution and the third solution through a triple-axial capillaries, thereby producing the concentric triple-layered microfiber.

According to some embodiments of the invention, the multi-layered microfiber or microtube comprises an exterior layer formed of a first polymeric solution, an intermediate layer formed of a second polymeric solution, and/or an interior layer formed of a third solution (a polymeric solution or a solution devoid of polymers).

In another embodiment, said first polymeric solution solidifies faster than said second polymeric solution. In another embodiment, said second polymeric solution solidifies faster than said third polymeric solution.

In some embodiments, the solvent selected for forming the exterior layer of the microfiber is an organic-based solvent and the solvent selected for forming the intermediate and/or internal layer of the microfiber is a solvent compatible for cell growth and viability.

As used herein, an “organic solvent” relate to a chemical class of compounds that are used routinely in commercial industries, which typically share a common structure (at least 1 carbon atom and 1 hydrogen atom), low molecular weight, lipophilicity, and volatility, and they exist in liquid form at room temperature. In some embodiments, the organic solvents are aliphatic-chain compounds, such as n-hexane. In other embodiments, the organic solvents are aromatic compounds with a 6-carbon ring, such as benzene or xylene. Aliphatics and aromatics may contain a substituted halogen element and may be referred to as halogenated hydrocarbons, such as perchloroethylene (PCE or PER), trichloroethylene (TCE), and carbon tetrachloride. Alcohols, ketones, glycols, esters, ethers, aldehydes, and pyridines are substitutions for a hydrogen group.

As used herein, a “solvent compatible for cell growth and viability” relates to a solvent which is substantially not-toxic for a cell. Non-limiting example of such solvents include water, glycerol-based solutions, and cell culture media.

In another embodiment, a solvent of said second polymeric solution is incapable of dissolving said first polymeric solution. In another embodiment, a solvent of said third polymeric solution is incapable of dissolving said second polymeric solution. In another embodiment, the second polymeric solution and said third solution comprise the same solvent.

As used herein the phrase “co-electrospinning” refers to a process in which at least three solutions (out of which at least two are polymeric solutions) are electrospun from co-axial capillaries (i.e., at least three capillary dispensers wherein one capillary is placed within an intermediate capillary which is placed within a third capillary while sharing a co-axial orientation) forming the spinneret. The capillary can be, for example, a syringe with a metal needle or a bath provided with one or more capillary apertures from which the polymeric solutions can be extruded, e.g., under the action of hydrostatic pressure, mechanical pressure, air pressure and/or high voltage.

The collector serves for collecting the electrospun element (e.g., the electrospun microfiber) thereupon. Such a collector can be a rotating collector or a static (non-rotating) collector. When a rotating collector is used, such a collector may have a cylindrical shape (e.g., a drum), however, the rotating collector can be also of a planar geometry (e.g., an horizontal disk). The spinneret is typically connected to a source of high voltage, such as of positive polarity, while the collector is grounded, thus forming an electrostatic field between the dispensing capillary (dispenser) and the collector. Alternatively, the spinneret can be grounded while the collector is connected to a source of high voltage, such as with negative polarity. As will be appreciated by one ordinarily skilled in the art, any of the above configurations establishes motion of a positively charged jet from the spinneret to the collector. Reverse polarity for establishing motions of a negatively charged jet from the spinneret to the collector are also contemplated.

For electrospinning, the first polymeric solution is injected into the outer capillary of the co-axial capillaries, the second polymeric solution is injected into the intermediate capillary of the co-axial capillaries, while the third solution is injected into the inner capillary of the co-axial capillaries. In one embodiment, such as in order to form a microtube (i.e., a hollow structure), the first polymeric solution (which is for forming the exterior layer of the microfiber) solidifies faster than the second polymeric solution (for forming the middle/intermediate layer of the microfiber), which in turn, solidifies faster than the third polymeric solution (for forming the interior layer of the microfiber). In another embodiment, the third polymeric solution (for forming the interior layer) is not a polymeric solution. In one embodiment, the interior layer is formed by electrospinning a suspension of cells and/or molecules of interest.

In another embodiment, the formation of a microfiber also requires that the solvent of the second polymeric solution be incapable of dissolving the first polymeric solution. In another embodiment, the solvent of the third polymeric solution is similar or identical to the solvent of the second polymeric solution In another embodiment, the solvent of the third polymeric solution be incapable of dissolving the second polymeric solution. The solidification rates of the first, second and third polymeric solutions may be critical for forming the microfiber. For example, for a microtube of about 100 the solidification of the first polymer (of the first polymeric solution) can be within about 30 milliseconds (ms), the solidification of the second polymer (of the second polymeric solution) can be within about 10-20 seconds, while the solidification of the third polymer (of the third polymeric solution) can be substantially longer than the second polymer. The solidification may be a result of polymerization rate and/or evaporation rate.

As used herein the phrase “polymeric solution” refers to a soluble polymer, i.e., a liquid medium containing one or more polymers, co-polymers or blends of polymers dissolved in a solvent or mixture of solvents. The polymer used by the invention can be a natural, synthetic, biocompatible and/or biodegradable polymer. 15

The phrase “synthetic polymer” refers to polymers that are not found in nature, even if the polymers are made from naturally occurring biomaterials. Examples include, but are not limited to, aliphatic polyesters, poly(amino acids), copoly(ether-esters), polyalkylenes, oxalates, polyamides, tyrosine derived polycarbonates, poly(iminocarbonates), polyorthoesters, polyoxaesters, polyamidoesters, polyoxaesters containing amine groups, poly(anhydrides), polyphosphazenes, and combinations thereof.

Suitable synthetic polymers for use by the invention can also include biosynthetic polymers based on sequences found in naturally occurring proteins such as those of collagen, elastin, thrombin, fibronectin, or mutant or synthetic derivatives thereof or, starches, poly(amino acids), poly(propylene fumarate), gelatin, alginate, pectin, fibrin, oxidized cellulose, chitin, chitosan, tropoelastin, hyaluronic acid, polyethylene, polyethylene terephthalate, poly(tetrafluoroethylene), polycarbonate, polypropylene and poly(vinyl alcohol), ribonucleic acids, deoxyribonucleic acids, polypeptides, proteins, polysaccharides, polynucleotides and combinations thereof.

The phrase “natural polymer” refers to polymers that are naturally occurring. Non-limiting examples of such polymers include, silk, collagen-based materials, chitosan, hyaluronic acid, albumin, fibrinogen, and alginate.

As used herein, the phrase “co-polymer” refers to a polymer of at least two chemically distinct monomers. Non-limiting examples of co-polymers include, polylactic acid (PLA)-polyethyleneglycol (PEG), polyethylene glycol terephthalate (PEGT)/polybutylene terephthalate (PBT), PLA-polyglycolic acid (PGA), PEG-polycaprolactone (PCL) and PCL-PLA.

As used herein, the phrase “blends of polymers” refers to the result of mixing two or more polymers together to create a new solution/composition with different physical properties.

The phrase “biocompatible polymer” refers to any polymer (synthetic or natural) which when in contact with cells, tissues or body fluid of an organism does not induce adverse effects such as immunological reactions and/or rejections, cellular death, and the like. A biocompatible polymer can also be a biodegradable polymer.

According to some embodiments of the invention, the first, the second and/or the third polymeric solutions are biocompatible.

Non-limiting examples of biocompatible polymers include polyesters (PE), PCL, PLA, PGA, PEG, polyvinyl alcohol, polyvinyl pyrrolidone, polytetrafluoroethylene (PTFE, teflon), polypropylene (PP), polyvinylchloride (PVC), polymethylmethacrylate (PMMA), polyamides, segmented polyurethane, polycarbonate-urethane and thermoplastic polyether urethane, silicone-polyether-urethane, silicone-polycarbonate-urethane collagen, PEG-DMA, alginate, hydroxyapatite and chitosan, blends and copolymers thereof.

The phrase “biodegradable polymer” refers to a synthetic or natural polymer which can be degraded (i.e., broken down) in a physiological environment such as by proteases or other enzymes produced by living organisms such as bacteria, fungi, plants and animals. Biodegradability depends on the availability of degradation substrates (i.e., biological materials or portion thereof which are part of the polymer), the presence of biodegrading materials (e.g., microorganisms, enzymes, proteins) and the availability of oxygen (for aerobic organisms, microorganisms or portions thereof), lack of oxygen (for anaerobic organisms, microorganisms or portions thereof) and/or other nutrients.

Examples of biodegradable polymers/materials include, but are not limited to, collagen (e.g., collagen I or IV), fibrin, hyaluronic acid, polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), polydioxanone (PDO), trimethylene carbonate (TMC), polyethyleneglycol (PEG), collagen, PEG-DMA, alginate, chitosan copolymers or mixtures thereof.

According to some embodiments, the polymeric solution can be made of one or more polymers, each can be a polymer or a co-polymer such as described hereinabove.

According to some embodiments of the invention, the polymeric solution is a mixture of at least one biocompatible polymer and a co-polymer (either biodegradable or non-biodegradable).

For certain applications one or more layers of the microfiber may comprise pores, thus creating a “breathing” tube. Methods of forming “breathing” microtube (i.e., microtubes with pores in the shell thereof) are described in PCT/IB2007/054001 to Zussman E., et al., which is fully incorporated herein by reference. Briefly, “breathing” fibers and/or tubes can be formed by the inclusion of a high percent (e.g., at least 80%) of a volatile component such as tetrahydrofuran (THF), chloroform, acetone, or trifluoroefhanol (TFE) in the first polymeric solution, and/or by the inclusion of a water-soluble polymer such as polyethylene glycol (PEG) in the first polymeric solution so that the first polymeric solution comprises a blend of polymers in which one is water-soluble and the other is water-insoluble (e.g., a blend of PEG and PCL).

Alternatively, “breathing” microtubes can be formed by inducing pores in one or more layers of the microfiber, after the completion of the electrospinning process, essentially as described in PCT WO 2006/106506, which is fully incorporated herein by reference, such as by passing an electrical spark or a heated puncturing element through the electrospun microfiber, or by using a pulsed or continuous laser beam through the electrospun microfiber.

According to some embodiments of the invention, the first polymeric solution comprises PEG for inducing pores in the exterior layer of the microfiber. For example, to generate pores greater (>) than 150 nm in diameter, the first polymeric solution may include about 4% PEG MW 35 kDa. Similarly, to generate pores smaller (<) 150 nm in diameter, the first polymeric solution may include about 2% PEG MW 6 kDa or less.

The microfiber can be designed for selective passage of certain molecules or particles. The passage through the exterior layer pores depends on the size and/or the electrical charge of the molecules/particles with respect to the geometry (length and radius), surface energy, electrical charge of the shell pores, and the viscosity and surface tension of the liquid containing the molecules/particles.

According to some embodiments of the invention, the porosity (i.e., the ratio of the volume of the pores to the volume of the mass) and pore size can control the release of the cell/molecules 30 from the microfiber. For example, a layer with pores larger than 1 μm in diameter (e.g., about 1-2 μm) can enable the release of a cell therethrough. In addition, increased porosity can result in a greater rate of release through the pores.

Alternatively, the microfiber can be made such that it prevents diffusion or passage of the cell, or any molecule therethrough (e.g., substantially devoid of pores, or with pores having a diameter which is smaller than the cell, or which exhibit a geometry which prevents passage of cells or membrane-coated particles therethrough).

EXAMPLES

Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA 10 techniques. Such techniques are thoroughly explained in the literature. See, for example, “Molecular Cloning: A laboratory Manual” Sambrook et al., (1989); “Current Protocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., “Current Protocols in Molecular Biology”, John Wiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide to Molecular Cloning”, John Wiley & Sons, New York (1988); Watson et al., “Recombinant DNA”, Scientific American Books, New York; Birren et al. (eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis, J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique” by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; “Current Protocols in Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J., eds. (1985); “Transcription and Translation” Hames, B. D., and Higgins S. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide to Molecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317, Academic Press; “PCR Protocols: A Guide To Methods And Applications”, Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategies for Protein Purification and Characterization—A Laboratory Course Manual” CSHL Press (1996); all of which are incorporated by reference. Other general references are provided throughout this document.

Materials and Methods:

The polymers PVDF-HFP (Mw=400,000 Da), PVP (Mw=1,300,000 Da) and PEG (Mw=6000 Da) and the solvents Dimethylforamide (DMF) and tetrahydrofuran (THF) were purchased from Sigma and were used without further purification. Glycerol was purchased from BioLab and was sterilized before use. De-ionized water with conductivity of <19 uS was used for aqueous solutions. Solutions for electrospinning were prepared by simple mixing for several hours. All experiments were conducted at room temperature and a relative humidity of 50-60%. The flow rates of all solutions were controlled by syringe pumps.

Table 1, represents different compositions used for fabricating the electrospun fibers using tri-axial spinnerets.

TABLE 1 Fibers compositions Internal Shell Intermediate layer layer/core 15% PVDF-HFP in DMF:THF in 15% PVP in water Yeast in water DMF:THF (1:2 v/v) + 2% PEG (150 mg/ml) 15% PVDF-HFP in DMF:THF in 15% PVP in water Yeast in water DMF:THF (1:2 v/v) + 4% PEG (150 mg/ml) 15% PVDF-HFP in DMF:THF in 15% PVP in water Yeast in water DMF:THF (1:2 v/v) + 6% PEG (150 mg/ml) 15% PVDF-HFP in DMF:THF in 15% PVP in water Yeast in water DMF:THF (1:2 v/v) + 8% PEG (150 mg/ml)

Encapsulation of yeast in fibers was successfully achieved using all compositions and spinning parameters given in the table above. The encapsulated yeast remained alive, active and proliferated inside the fibers utilizing the internal space of the fiber/tube and expand until full space was occupied with a densely packed cells.

Use of the tri-axial spinneret enabled to introduce a larger amount of cells (about 10 times more) during the electrospinning process. This shortened the period of the post spinning recovery period and the fibers were subjected to the actual medium sooner.

Descriptions of embodiments of the invention in the present application are provided by way of example and are not intended to limit the scope of the invention. The described embodiments comprise different features, not all of which are required in all embodiments of the invention. Some embodiments utilize only some of the features or possible combinations of the features. Variations of embodiments of the invention that are described, and embodiments of the invention comprising different combinations of features noted in the described embodiments, will occur to persons of the art. The scope of the invention is limited only by the claims. 

1. A concentric multi-layered electrospun microfiber comprising an exterior layer, an interior layer, and an intermediate layer residing between the exterior layer and the interior layer, said intermediate layer comprises one or more water soluble polymers, and said interior layer comprises cells and/or molecules of interest.
 2. The concentric multi-layered electrospun microfiber of claim 1, wherein interior layer further comprises water soluble polymers or oligomers.
 3. The microfiber of claim 1 being a triple-layer microfiber.
 4. The microfiber of claim 1, wherein said microfiber is hollow.
 5. The microfiber of claim 1, wherein said interior layer, intermediate layer and/or interior layer are substantially porous layers.
 6. The microfiber of claim 1, wherein said water soluble polymers of said intermediate layer are selected from natural polymers, synthetic polymers or combinations thereof.
 7. The microfiber of claim 1, wherein said exterior layer comprises one or more organic soluble polymers.
 8. The microfiber of claim 7, wherein said organic soluble polymer of said exterior layer is selected from the group consisting of: poly(vinylidenefluoride), poly (e-caprolactone) (PCL), polyamide, poly(siloxane), poly(silicone), poly(ethylene), poly(2-hydroxy ethylmethacrylate), poly(methyl methacrylate), poly(vinyl acetate), polyacrylamide, poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polylactide, polyglycolide, poly(lactide-co-glycolide), polyanhydride, polyorthoester, poly(carbonate), poly(acrylo nitrile), polyaniline, polyvinyl carbazole, polystyrene, poly(vinyl phenol), polyhydroxyacid, poly(caprolactone), polyanhydride, polyhydroxyalkanoate, polyurethane, collagen, cellulose, and combination/copolymers thereof.
 9. The microfiber of claim 7, wherein said organic-soluble polymer is poly(vinylidenefluoride-co-hexafluor-opropylene) (PVDF-HFP).
 10. The microfiber of claim 1, wherein said exterior layer comprises any one of water-soluble polymers and antifouling agents.
 11. The microfiber of claim 10, wherein said exterior layer comprises water soluble polymers selected from the group consisting of: poly(vinyl pyrrolidone), poly(N-vinyl pyrrolidone), poly(vinyl alcohol), poly(acrylic acid), poly(ethylene glycol), poly(ethylene oxide), albumin, alginate, chitosan, starch, hyaluronic acid, albumin, alginate, chitosan, starch, polypetides and combination/ copolymers thereof.
 12. (canceled)
 13. The microfiber of claim 1, wherein said water soluble polymers of said intermediate layer are selected from the group consisting of poly(acrylic acid), poly(vinyl pyrrolidone), poly(N-vinyl pyrrolidone), poly(vinyl alcohol), poly(acrylic acid) poly(ethylene glycol), poly(methacrylic acid), poly(ethylene oxide), polyhydroxyacid, alginate, starch, chitosan, dextran, albumin, chitosan, hyaluronic acid, polyppertides and combination/copolymers thereof.
 14. The microfiber of claim 2, wherein said water soluble polymers and/or oligomers of said interior layer are selected from natural and synthetic polymers and/or oligomers, or combinations thereof.
 15. The microfiber of claim 14, wherein any one of: (i) said water soluble synthetic polymer is selected from the group consisting of PVA, PVP, PEO, PAA, polystyrene sulfonate and combinations or copolymers thereof, (ii) said water soluble natural polymer is selected from the group consisting of polysaccharides (e.g. alginate), starch, hyaluronic acid, alginate, dextran, chitosan and combinations or copolymers thereof; and (iii) said oligomer is selected from the group consisting of chitosan oligomeric derivatives, and saccharides oligomers.
 16. (canceled)
 17. (canceled)
 18. The microfiber of claim 1, wherein the cell concentration within said internal layer is at least 10 OD.
 19. The microfiber of claim 1, wherein said interior layer further comprises media for maintaining and/or supporting said cell viability and/or activity.
 20. The microfiber of claim 1, wherein said molecules of interest are selected from hormones, growth factors, enzymes, nutrients and shock resistance molecules.
 21. The microfiber of claim 1, wherein any one of said exterior layer, intermediate layer, or internal layer has a thickness of about 5 nm to 10 micrometer, optionally wherein said interior layer has a diameter of at most 50 micrometer.
 22. (canceled)
 23. (canceled)
 24. (canceled)
 25. A method for producing a concentric triple-layered microfiber, the method comprising: providing a first polymeric solution for forming an exterior layer of the microfiber, a second polymeric solution for forming an intermediate layer of the microfiber, and a third solution for forming an interior layer or a core of the microfiber, said third solution comprises cells; and co-electrospinning the first polymeric solution, second polymeric solution and the third solution through a triple-axial capillaries, thereby producing the concentric triple-layered microfiber.
 26. The method of claim 25, wherein any one of (i) the first polymeric solution solidifies faster than the second polymeric solution; (ii) the second polymeric solution solidifies faster than the third solution; (iii) the second polymeric solution and said third solution comprise the same solvent; (iv) the second polymeric solution comprises a solvent which is incapable of dissolving the first polymeric solution; (v) any combination thereof.
 27. (canceled)
 28. (canceled)
 29. (canceled) 